Summary
We present a gel-free proteomics procedure for the specific isolation of phosphorylated peptides from whole proteome digests. Central is the use of diagonal, reverse-phase chromatography which consists of two consecutive reverse-phase peptide separations with a modification step in between. The latter alters the column retention of affected peptides, thereby allowing their specific isolation from the bulk of nonaffected peptides. To isolate phosphopeptides from complex mixtures, this modification step is a dephosphorylation reaction using a cocktail of broad-spectrum phosphatases. Upon dephosphorylation, peptides undergo a hydrophobic shift and are thereby sorted from in vivo nonphosphorylated peptides. To increase the overall yield of phosphopeptides, a pre-enrichment step was found necessary and to further distinguish true ex-phosphorylated peptides from nonphosphorylated peptides sorted artificially, differential isotope labeling was introduced. The complete COFRADIC sorting procedure is described here.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Manning, G., Whyte, D. B., Martinez, R., Hunter, T., and Sudarsanam, S. (2002) The protein kinase complement of the human genome. Science 298, 1912–34.
Chu, G., Egnaczyk, G. F., Zhao, W., Jo, S. H., Fan, G. C., Maggio, J. E., Xiao, R. P., and Kranias, E. G. (2004) Phosphoproteome analysis of cardiomyocytes subjected to beta-adrenergic stimulation: identification and characterization of a cardiac heat shock protein p20. Circ Res 94, 184–93.
Maguire, P. B., Wynne, K. J., Harney, D. F., O'Donoghue, N. M., Stephens, G., and Fitzgerald, D. J. (2002) Identification of the phosphotyrosine proteome from thrombin activated platelets. Proteomics 2, 642–8.
Martin, K., Steinberg, T. H., Goodman, T., Schulenberg, B., Kilgore, J. A., Gee, K. R., Beechem, J. M., and Patton, W. F. (2003) Strategies and solid-phase formats for the analysis of protein and peptide phosphorylation employing a novel fluorescent phosphorylation sensor dye. Comb Chem High Throughput Screen 6, 331–9.
Gevaert, K., Van Damme, P., Ghesquière, B., Impens, F., Martens, L., Helsens, K., and Vandekerckhove, J. (2007) A la carte pro-teomics with an emphasis on gel-free techniques. Proteomics 7, 2698–718.
Wu, C. C., MacCoss, M. J., Howell, K. E., and Yates, J. R., 3rd (2003) A method for the comprehensive proteomic analysis of membrane proteins. Nat Biotechnol 21, 532–8.
Ficarro, S. B., McCleland, M. L., Stukenberg, P. T., Burke, D. J., Ross, M. M., Shabanowitz, J., Hunt, D. F., and White, F. M. (2002) Phos-phoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat Biotechnol 20, 301–5.
Pinkse, M. W., Uitto, P. M., Hilhorst, M. J., Ooms, B., and Heck, A. J. (2004) Selective isolation at the femtomole level of phos-phopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal Chem 76, 3935–43.
Beausoleil, S. A., Jedrychowski, M., Schwartz, D., Elias, J. E., Villen, J., Li, J., Cohn, M. A., Cantley, L. C., and Gygi, S. P. (2004) Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci USA 101, 12130–5.
Oda, Y., Nagasu, T., and Chait, B. T. (2001) Enrichment analysis of phosphorylated proteins as a tool for probing the phosphopro-teome. Nat Biotechnol 19, 379–82.
Zhou, H., Watts, J. D., and Aebersold, R. (2001) A systematic approach to the analysis of protein phosphorylation. Nat Biotechnol 19, 375–8.
Brown, J. R., and Hartley, B. S. (1966) Location of disulphide bridges by diagonal paper electrophoresis. The disulphide bridges of bovine chymotrypsinogen A. Biochem J 101, 214–28.
Cruickshank, W. H., Malchy, B. L., and Kaplan, H. (1974) Diagonal chromatography for the selective purification of tyrosyl pep-tides. Can J Biochem 52, 1013–7.
Gevaert, K., Van Damme, J., Goethals, M., Thomas, G. R., Hoorelbeke, B., Demol, H., Martens, L., Puype, M., Staes, A., and Vandekerckhove, J. (2002) Chromatographic isolation of methionine-containing peptides for gel-free proteome analysis: identification of more than 800 Escherichia coli proteins. Mol Cell Proteomics 1, 896–903.
Van Damme, P., Martens, L., Van Damme, J., Hugelier, K., Staes, A., Vandekerckhove, J., and Gevaert, K. (2005) Caspase-specific and nonspecific in vivo protein processing during Fas-induced apoptosis. Nat Methods 2, 771–7.
Ghesquière, B., Van Damme, J., Martens, L., Vandekerckhove, J., and Gevaert, K. (2006) Proteome-wide characterization of N-glyco-sylation events by diagonal chromatography. J Proteome Res 5, 2438–47.
Ghesquière, B., Buyl, L., Demol, H., Damme, J. V., Staes, A., Timmerman, E., Vandeker-ckhove, J., and Gevaert, K. (2007) A new approach for mapping sialylated N-glycosites in serum proteomes. J Proteome Res 6, 4304–12.
Gevaert, K., Van Damme, P., Ghesquière, B., and Vandekerckhove, J. (2006) Protein processing and other modifications analyzed by diagonal peptide chromatography. Biochim Biophys Acta 1764, 1801–10.
Gevaert, K., Impens, F., Van Damme, P., Ghesquiere, B., Hanoulle, X., and Vandeker-ckhove, J. (2007) Applications of diagonal chromatography for proteome-wide characterization of protein modifications and activity-based analyses. Febs J 274, 6277–89.
Gevaert, K., Staes, A., Van Damme, J., De Groot, S., Hugelier, K., Demol, H., Martens, L., Goethals, M., and Vandekerckhove, J. (2005) Global phosphoproteome analysis on human HepG2 hepatocytes using reversed-phase diagonal LC. Proteomics 5, 3589–99.
Staes, A., Demol, H., Van Damme, J., Martens, L., Vandekerckhove, J., and Gevaert, K. (2004) Global differential non-gel pro-teomics by quantitative and stable labeling of tryptic peptides with oxygen-18. J Proteome Res 3, 786–91.
Liu, P., Feasley, C. L., and Regnier, F. E. (2004) Optimization of diagonal chromatog-raphy for recognizing post-translational modifications. J Chromatogr A 1047, 221–7.
Acknowledgments
The authors acknowledge support by research grants from the Fund for Scientific Research – Flanders (Belgium) (project numbers G.0156.05, G.0077.06, and G.0042.07), the Concerted Research Actions (project BOF07/GOA/012) from the Ghent University, the Inter University Attraction Poles (IUAP06), and the European Union Interaction Proteome (6th Framework Program).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Gevaert, K., Vandekerckhove, J. (2009). Reverse-Phase Diagonal Chromatography for Phosphoproteome Research. In: Graauw, M.d. (eds) Phospho-Proteomics. Methods in Molecular Biology™, vol 527. Humana Press. https://doi.org/10.1007/978-1-60327-834-8_16
Download citation
DOI: https://doi.org/10.1007/978-1-60327-834-8_16
Publisher Name: Humana Press
Print ISBN: 978-1-60327-833-1
Online ISBN: 978-1-60327-834-8
eBook Packages: Springer Protocols